The prion diseases like Alzheimer's disease (AD) are age-dependent, degenerative neurological disorders. CNS degeneration caused by prions is the most well understood of the disorders of delayed onset and they are the focus of this proposal. Much evidence argues that prion diseases are disorders of protein conformation. Investigation directed toward elucidating the molecular mechanism of conversion of the cellular prion protein) (PrP/c) into the scrapie isoform (PrP/sc) are proposed. Using molecular genetic approaches, we plan to extend recent findings showing that an artificial mutation (Ala>Val) in the first putative region of secondary structure in PrP produces neurodegeneration in transgenic (Tg) mice was found to adopt spontaneously a conformation which is high in beta-sheet and similar to that of PrP/sc. Additionally, we found that two large segments of PrP can be deleted with the resulting protein of 106 residues being converted into a soluble PrP/sc-like molecule. We propose to create additional amino acid substitutions and deletion constructs. We shall determine the NMR structures of soluble, full length wild-type (wt) and mutant PrPs that have been expressed in E. coli (Project 2). In parallel, we plan to determine the structures of the recombinant PrPs expressed both in E. coli and Tg mice by X-ray crystallography (Project 3). A third approach to determining the structures of PrP/c and PrP/sc utilizes batteries of recombinant antibodies (Project 4). Such antibodies have been produced and used to identify portions of the PrP molecule that refold during conversion of PrP/c into PrP/sc. The diverse skills, talents and backgrounds of the investigators in the proposed program offer an unusual opportunity to define the molecular mechanisms responsible for neurodegeneration. Elucidation of the mechanisms by which brain cells cease to function and die in prion disease after a long delay may offer new approaches to elucidating the etiologies of more prevalent neurodegenerative disorders afflicting other people, including AD.
